Historically maize (corn) has been used as a source of food for human and animal consumption. Even today maize supplies about twenty percent of the world's calories. Any environmental stress factor that affects a large proportion of the maize growing regions can have a substantial impact on the quantity of maize annually available for consumption. Thus reduction of the sensitivity associated with HpH is understandably of great importance since approximately 10-12 million corn growing acres are affected by high pH (basic) soil. Therefore the development of high pH soil tolerant maize is the subject of interest to many commercial corn breeding programs. It is known in the industry that HpH soil has detrimental effects on the hybrids yield potential. Professors Paul Nordquist and Gary Hergert, Professors of Agronomy, did a corn pH tolerance study in 1992 on corn hybrids in calcareous and alkali soils and the following FIG. 1 gives their results. FIG. 1 clearly evidences the loss in yield associated with calcareous soil.
High pH (basic) soils are prevalent throughout the North Central and Western corn belt. The HpH (greater than 7.5 pH) of the soil arises due to a predominantly calcareous bed-rock, combined with low rainfall and relatively low organic matter in the soil. The high pH results in maize plants having an induced iron (Fe) deficiency syndrome. Most corn grown in high pH soil has a syndrome that is expressed visually in sensitive genotypes as stunting, yellowing of leaves, reduced dry matter accumulation, sterility or severely delayed pollen shed, poor silk development, and reduced yield. The HpH syndrome appears to be a result of the maize plant's inability to utilize the necessary nutrients from HpH soil particularly iron.
Although when tested many high pH soils appear to have the basic nutrients necessary for the growth and development of the maize plant. Even in soils that do not have all of the necessary nutrients, addition of the nutrients to high pH soils does not appear to result in a reduction of the high pH syndrome. Suggesting that it is not the availability of the nutrients per se but the ability of the plant to assimilate the nutrient under conditions of HpH.
Although iron (Fe) is a major component of most soils, it exists as silicates, oxides and hydroxides, and is sparingly soluble in well aerated soils, even at low pH. It has been demonstrated that Fe.sup.3+ (the form of iron used by plants), has exceedingly low solubility in soils of pH 7.0 and above; and that Fe solubility decreases by three orders of magnitude for every unit increase in pH. The syndrome of high pH stress can be compounded by bicarbonates in the soil solution or irrigation water, reduced soil aeration, cool temperatures and high light intensities.
To date there are some fairly high pH tolerant maize hybrids that have been developed by traditional breeding methods. Unfortunately, it is difficult to transfer this tolerance to new inbreds and additionally it is difficult to transfer it to new hybrid products. In fact, it is quite common to see some poor agronomic traits and loss of tolerance associated with moving the tolerance trait from inbred to inbred.
Heretofore, few if any, truly agronomically desirable varieties of the corn in the hybrids have tolerance to high pH soil and also have the necessary agronomic traits for commercial production. Given that pursuant to this invention it has been discovered four genes, three of high significance control the maize plant's response to high pH, a progeny containing these genes, one of which is recessive, within a genome is expected to be a very rare occurrence. The presence of the four newly discovered alleles in the same genome is a very rare occurrence.
One of the fundamental principles of maize breeding is a production of a hybrid having the desired mix of traits by the combination of two inbreds. Getting the correct mix of traits in two inbreds to produce a hybrid especially when traits are not directly associated with a phenotype characteristic of a plant can be difficult. To produce improved hybrids, there is an ongoing development of new inbreds. An inbred is a plant which has become homozygous at almost all loci. There are two primary germplasm sources for producing new inbreds. One source is germplasm that has been genetically engineered; the second source is an adapted or an unadapted germplasm.
In a conventional breeding program, pedigree breeding and recurrent selection breeding methods are employed to develop new inbred lines with desired resistant traits. Maize breeding programs attempt to develop these inbred lines by self-pollinating plants and selecting the desirable plants from the populations. An inbred produces a uniform population of hybrid plants when crossed with a second homozygous line, i.e., inbred. Inbreds tend to have poor vigor and low yield; however, the progeny of an inbred cross usually evidences vigor. The progeny of a cross between two inbreds is often identified as an F.sub.1 hybrid. The resultant F.sub.1 hybrids which may be heterozygous at a number of loci, are evaluated to determine whether or not they show the tolerance trait and agronomically important and desirable traits. Identification of desirable agronomic traits has typically been done by breeders' expertise. A plant breeder identifies a desired trait for the area in which his plants are to be grown and selects inbreds which appear to pass the desirable trait or traits on to the hybrid.
Conventional plant breeders rely on phenotypic traits of the inbreds for selection purposes. Modern plant breeding technology looks at the genotypic material (chromosomes) for plant breeding purposes. One method of looking at plant genotypes is to use Restriction Fragment Length Polymorphisms (RFLPs) which provide a method for identifying the chromosomal regions which affect the agronomic traits in the plant genome which the plant breeder is attempting to introgress into the inbred line for ultimate expression in the hybrid.
RFLPs can be used to identify chromosomal regions in maize which is a ten chromosome plant. Each chromosome has a short arm with a distal and proximal end and a long arm having a distal and proximal end. Between the short arm proximal end and long arm proximal end is a centromere. Each chromosome is made up of strands of the deoxyribonucleic acid (DNA) molecule which has a specific nucleic acid sequence. Selected restriction endonucleases will identify a specific base sequence and cleave the DNA molecule wherever this sequence occurs. The resultant cleaved portions are called restriction fragments. These restriction fragments can be separated by size by electrophoresis through agarose gels.
The DNA of two individual maize plants will differ in sequence at a variety of sites. Because of this difference, restriction endonucleases may cleave an individual's DNA at a different site or location than the other individual's DNA. A polymorphism in the length of restriction fragments is produced when the fragments of the two individuals have different lengths. A polymorphism is detected by placing the fragments on an agarose gel electrophoresis and allowing them to separate by size over distance. A southern blot is then used. The fragments of the DNA are physically transferred on to a membrane, then nucleic acid hybridization detects the sequences by hybridization of the single strand of DNA (probe) on the southern blot. The nucleic acid reforms double stranded DNA. A radio labelled probe is used to detect a particular (DNA) sequence. One method is to use a labelled probe such that the DNA fragment will be identifiable through autoradiography techniques.
A variety of maize genes have been mapped and identified using RFLPs. Certain polymorphisms (molecular markers) are used to identify chromosomal areas associated with certain traits. A large number of molecular markers including RFLPs have been applied to the maize genome and a detailed maize genetic linkage map that can be used to localize important genes has been constructed.
A variety of traits have been identified by RFLPs; for example, P1 pericarp color has been linked to UMC185 (P1) on the short arm of chromosome one of the maize plant. Probes BNL6.29 and UMC85 on chromosome six of the maize plant have been identified with Maize Dwarf Mosaic Virus (MDMV) strain A resistance in maize. Likewise, a variety of other traits have been genetically identified and placed on the maize genetic linkage map.
It would appear that once a desired trait is recognized and the gene chromosome region expressing that trait is located between flanking probes in a maize plant by the use of RFLPs, that the trait should be readily introgressed into an inbred line. Unfortunately, it is not easy to recognize the desired gene location and although RFLPs are a tool which can be employed to help identify the chromosomal region to which the trait appears to be linked, RFLPs are not a solution in and of themselves. RFLPs are simply a tool of identification. It should be noted that the chromosomal regions associated with high pH tolerance have not been previously mapped or identified using probes.
Almost nothing has hitherto been known about the genes responsible for tolerance to high pH. The number of genes involved, their action, where they are located on the maize chromosome have not been identified. There is a need for the identification of the location of genes associated with tolerance to high pH which permits their tracking when introgressed into new plants through traditional breeding. There also remains a need for a method of transferring tolerance to high pH soil to a corn inbred that has desirable agronomic traits. There remains a need for tolerant high pH inbreds and hybrids.